Project Summary Zebra finches (Taeniopygia guttata) are an important non-traditional avian model organism that have made major contributions to a range of questions with important implications for human health and disease. Funded by several NIH Institutes (e.g. NINDS, NIDCD, NIMH, NIGMS, NHGRI, and NIDA), studies in finches have addressed such topics as vocal communication, endocrine modulation of reproductive and social behaviors, sex steroids actions in brain and behavior, sex dimorphisms, the role of sleep in learning, motor coding and rhythms, mechanisms of hearing function, the dynamics of adult neurogenesis and neuronal replacement, and the role of endocannabinoids in cell signaling and addiction, among other themes. Finches are also the prime model organism for studying the neuronal and genetic basis of vocal learning, a trait that provides the basis of human speech and language learning and that is absent or rudimentary in rodents and monkeys. A major bottleneck to finch studies, however, has been the lack of high efficiency tools for gene manipulation, which has limited the ability to establish causal links between genes and phenotypes of interest in these birds. VSV- G-pseudotyped lentiviruses (VSV-G-LVs) are routinely used in gene manipulation studies because they readily transduce a broad range of tissues and organisms, however they show very low transduction in finches. We have recently found that the receptor for VSV-G-LVs, namely the low density lipoprotein receptor (LDLR), is a pseudogene in finches, providing a likely explanation for the low LV transduction. We have also obtained evidence that finches have evolved unique features of cholesterol metabolism that may compensate for this gene loss, consistent with a central and conserved role for LDLR in cholesterol uptake, as well as deleterious effects on health when this gene is disrupted in humans (e.g. increased risk for coronary disease, genetic hypercholesterolemia). Importantly, we have found that expressing human LDLR (hLDLR) in cultured finch cells markedly boosts VSV-G-LVs transduction, pointing to a plausible solution for the low VSV-G-LV efficacy. In this exploratory R21, we propose to: SA1) Determine the effectiveness of LDLR constructs for VSV-G- LV transduction. We will use an in vitro assay with cultured finch cells to examine how hLDLR-based constructs that vary in the domains for viral binding or receptor recycling affect VSV-G-LV transduction; and SA2) Generate and characterize zebra finch transgenic lines harboring a functional hLDLR. We will use established protocols to generate and characterize transgenic finches that constitutively and ubiquitously express LDLR constructs engineered to maximize VSV-G-LV transduction. We predict that these transgenic lines will show high VSV-G-LV transduction across multiple tissues, making them important resources for future gene manipulation and CRISPR-based gene editing applications that address a wide range of biomedical questions. They will also serve as novel tools for better understanding the genetics and evolution of LV infectivity and cholesterol metabolism in vertebrates.